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Abstract

Background

In-situ hybridisation studies demonstrate that Notch receptors and ligands are expressed
in granulosa cells (GCs) and in the theca layer vasculature of growing follicles.
Notch signaling involves cell-to-cell interaction mediated by transmembrane receptors
and ligands. This signaling pathway may represent a novel intraovarian regulator of
gonadotropin-dependent follicular development to the preovulatory stage. We hypothesized
that blocking Notch pathways would disrupt follicular maturation in the mouse ovary.

Methods

Hypophysectomized CD21 female mice were administered pregnant mare serum gonadotropin
(PMSG) for 3 days to stimulate follicular development. In one experiment, a pan-notch
inhibitor, compound E, was initiated 2 days prior to and throughout stimulation (n = 10),
while in a second experiment, a humanized phage Dll4 blocking antibody, YW152F, was used (n = 5). After sacrifice, ovarian histology, serum estradiol levels and
uterine weights were compared to controls. The ovarian morphology was evaluated with
hematoxylin/eosin staining and immunohistochemistry was performed for Notch1, Notch2,
Notch3, Notch4, Jagged1, Dll4, platelet endothelial cell adhesion molecule (PECAM)
and alpha-smooth muscle actin (α-SMA) detection.

Results

We localized specific Notch ligands and receptors in the following structures: Dll4
is specific to theca layer endothelial cells (ECs); Notch1/Notch4 and Jagged1 are
expressed in theca layer ECs and vascular smooth muscle cells (VSMCs), whereas Notch3
is restricted to VSMCs; Notch2 is expressed mostly on GCs of small follicles. Administration
of a pan-Notch inhibitor, compound E, inhibits follicular development to the preovulatory
stage (8.5 preovulatory follicles in treatment vs. 3.4 preovulatory follicles in control,
p < 0.01; average number per ovary) with significant secondary effects on ovarian
and uterine weight and estradiol secretion in a setting of uninhibited vascular proliferation,
but disorganized appearance of ECs and VSMCs. Inhibition of endothelial Notch1 function
through the inactivation of its ligand Dll4 with the blocking antibody YW152F induces
mild disorganisation of follicular vasculature, but has no significant effect on gonadotropin-dependent
folliculogenesis.

Conclusions

Our experiments suggest that the complete blockage of the Notch signaling pathway
with compound E impairs folliculogenesis and induces disruption of gonadotropin stimulated
angiogenesis. It seems the mechanism involves Notch1 and Notch3, specifically, causing
the improper assembly of ECs and VSMCs in the theca layer, although the potential
role of non-angiogenic Notch signaling, such as Jagged2 to Notch2 in GCs, remains
to be elucidated.

Keywords:

Background

Major advances have been made in identifying and characterizing the role of intraovarian
regulators such as insulin growth factor (IGF), epidermal growth factor (EGF), vascular
endothelial growth factor (VEGF), transforming growth factors, anti-Müllerian hormone,
bone morphogenetic protein with respect to gonadotropin-dependent follicular development
[1]. Despite these advances, our understanding of how folliculogenesis is regulated is
far from complete, which suggests the existence of other unidentified intraovarian
regulators. In-situ hybridisation studies have shown that vascular and non-vascular
components of the Notch pathway are localized to specific structures in the ovary
[2,3]. For example m-RNA of Notch2, Notch3, and Jagged2 as well as downstream targets of
Notch are highly expressed in the granulosa cells (GCs) of developing follicles [2]. Vascular Notch m-RNA (Notch1 and Notch4) was detected on blood vessels in the theca
layer of growing follicles [2], a finding later validated by immunofluorescent studies [4]. Notch1 and the Notch ligand Jagged1 can be detected on ECs as well as vascular smooth
muscle cells (VSMCs) [4]. The Notch ligands Dll1 and Dll3 are absent in the ovary [2], whereas the Notch1 ligand Dll4 was detected by in-situ hybridisation in ovarian
vasculature [2,5]. Results derived from expression analysis suggest that Notch is a novel intraovarian
regulator, which regulates folliculogenesis through vascular and non-vascular mechanisms
[2,6]. It should be noted that Notch would be unique among intraovarian regulators as Notch
ligands and receptors are single-pass transmembrane proteins, requiring a juxtacrine
(or contact-dependent) signaling mechanism [3,7,8].

We hypothesized that blocking Notch pathways would disrupt in-vivo folliculogenesis in our mouse model by affecting vascular and non-vascular pathways.
This would confirm the effects on folliculogenesis described in vitro[3], but also evaluate vascular growth disruption surrounding maturing follicles. We
used a mouse model to perform functional studies using a pan-Notch inhibitor, compound
E, as well as a blocking antibody (BAb) against the Notch1 ligand Dll4, located exclusively
on endothelial cells (ECs). As in-situ hybridisation studies can be discrepant with
localisation of the corresponding protein, we performed immunofluorescence with antibodies
to Notch2, Notch3, and Dll4.

Methods

The study was reviewed and approved by the Institutional Review Board and the Institutional
Animal Care Committee of the Columbia University Medical Center.

Animal model

CD21 female mice (Charles River, Wilmington, MA, USA), hypophysectomized before 22
days of life, were used for all experiments. Insignificant weight gain (< 2 g over
8 days after arrival) and low estrogenic state vaginal smears verified that the surgery
had been successful in arresting follicular growth at the advanced preantral stage
due to the absence of pituitary gonadotropin secretions [9].

Experiment 2: Treatment group animals (n = 5) were injected with the Genentech anti-Dll4
blocking antibody (BAb) YW152F (10 mg kg-1) [11] 1 day prior and 1 day after PMSG administration. The antibodies were diluted in a
total volume of 170 μL DMSO and the solution was administered i.p. Control animals
(n = 5) were injected with human IgG using the same dose and regimen. Performance
of the experiment was otherwise done as described in experiment 1.

Histology

All animals were sacrificed 5 days after the initiation of compound E or DMSO treatment
and 4 days after anti-Dll4 BAb YW152F administration. Both ovaries and the uterus
were removed and weighed. Ovaries were embedded in optimal cutting temperature (OCT)
medium (Thermo Fisher Scientific, Waltham, MA, USA), flash frozen and stored at -80°C.
One whole ovary was sectioned serially (12 μm), and each section was stained with
hematoxylin/eosin (H&E) to count the total number of gonadotropin-dependent preovulatory
follicles per ovary as described previously [9]. Sections of the contra-lateral ovary (7 μm) of each mouse were used for specific
immunohistochemistry [9]. A piece of small intestine was flushed gently with cold phosphate buffered saline
(PBS) followed by a flush of formalin. The tissue was then fixed in formalin at 21°C
for 16 h. Intestinal sections were stained with periodic acid-Shiff (PAS) staining
(Sigma-Aldrich, St. Louis, MO, USA) in order to detect goblet cells, since Notch γ-secretase
inhibition turns proliferative cells in intestinal crypts into goblet cells [10]. An increase in the number of goblet cells in the treatment group over control group
served as a positive control demonstrating that compound E is active. Intestines from
animals of experiment 2 were not stained for goblet cells as they are not affected
by anti-Dll4 antibodies [11]. Blood was obtained through cardiopuncture for the measurement of estradiol (E2)
levels as described previously [9].

Data analysis

For each animal, all H&E sections from one ovary were evaluated to count the total
number of preovulatory follicles per ovary as previously described [9]. Statistical analysis was performed using the Statistical Package for Social Science
version 15 (SPSS, Inc., Chicago, IL, USA). Data are expressed as mean ± standard error
(SE). We used an unpaired t-test to compare sample means with statistical significance
defined as p < 0.05.

Results

Immunofluorescent studies

Notch2 is expressed in GCs of small follicles; Notch3 and Dll4 are expressed in follicular
vasculature.

Using immunofluorescent analysis, we found that Notch2 is expressed in GCs of secondary
follicles and sporadically in GCs of preovulatory follicles, but is absent in the
peripheral theca layer (Figure 1). Notch3 expression is largely restricted to VSMCs located in the theca layer of
growing follicles and in interstitial tissue (Figure 2). No evidence of Notch3 expression was seen in follicular GCs. The Notch1/Notch4
ligand, Dll4, is expressed exclusively in ECs in the follicular theca layer vasculature
and ovarian interstitial tissue during the follicular phase (Figure 3). Previously reported vascular expression patterns of Notch1 (ECs and VSMCs), Notch4
(ECs and VSMCs), and Jagged1 (ECs and VSMCs) [4] were confirmed (data not shown).

Figure 1.Notch2 (red) in developing follicles. Intense staining in secondary follicles and sporadic red staining in tertiary follicles
with complete absence of staining in the peripheral theca layer of the depicted 2
large follicles (10× magnification, DAPI counterstaining in blue).

Functional studies

Compound E

The pan-Notch inhibitor, compound E, inhibits gonadotropin-dependent follicle development
to the preovulatory stage.

Administration of the pan-Notch inhibitor, compound E [12], induced a decrease in the number of follicles maturing to the preovulatory stage
when compared to control after gonadotropin stimulation: control group: 8.5 ± 0.7
(mean ± SEM); treatment group: 3.8 ± 0.8 (p < 0.01) (Figure 4, Table 1). In addition, the growing follicles in the treatment group were smaller in size
and irregular in shape (Figure 4). The mean plasma E2 level in the control group was 83.4 ± 6.5 pg/mL, whereas in
the treatment group it was 29.3 ± 5.2 pg/mL (p < 0.01). Consistent with a lower number
of follicles in the ovaries in the treatment group, the mean ovarian weight was significantly
lower in the animals treated with compound E (control 5.8 ± 0.8 mg versus treatment
3.5 ± 0.3 mg; p < 0.01). Uterine weight, reflecting estrogen activity, was also lower
in the treatment group (control 42.9 ± 6.7 mg versus treatment 32.3 ± 2.3 mg; p < 0.05),
as shown in Table 1.

The density of VSMCs expressing alpha-smooth muscle actin (α-SMA) in the theca layer
of follicles and interstitial tissue of compound E treated animals (Figure 5A-D: Notch3 and α-SMA) was increased when compared to control. VSMCs had a very disorganized
appearance with increased vascular thickness when compared to control (Figure 5A). VSMCs continuity surrounding individual follicles was often disrupted (Figure 5B). A similar pattern of disorganization was seen for endothelial cells with an increase
in density in the treatment group when compared to control (Figure 5E and 5F). Double staining for PECAM and α-SMA showed mostly an organized pattern of overlap
in the control group (Figure 5E) as described previously [4]. In contrast to the treatment group, many endothelial cells (green) are devoid of
adjacent VSMCs (Figure 5F). Proliferation of non-GCs, representing mostly dividing endothelial cells and VSMCs,
was detected (Figure 6) demonstrating that compound E did not stop angiogenic proliferation. When comparing
proliferation to the control group, it appears that vascular proliferation might even
be increased in the treatment group (Figure 6), possibly explaining the increase in vascular density seen in compound E treated
ovaries. Therefore, inhibition of gonadotropin-dependent follicle growth occurs in
a setting of ongoing angiogenesis.

Table 2.Effect of Dll4 blocking antibody YW152F on total pre-ovulatory follicle number, mean
ovarian and uterine weights and serum estradiol levels when compared to control animals

Discussion

To understand the possible treatment effects of interrupting Notch signaling with
compound E or an anti-Dll4 BAb on gonadotropin-dependent folliculogenesis, one has
to have a good understanding of where these molecules are expressed within the follicles.
Complementary analysis of the expression of the Notch family proteins combined with
preexisting data [2,4,5] has allowed us to obtain a better idea about which type of cell-to-cell Notch signalling
occurs in growing follicles. We demonstrated that Notch3 is expressed exclusively
in vascular smooth muscle cells (VSMCs), which are adjacent to theca layer endothelial
cells [4]. The presence of Notch3 together with Notch1 [4] on VSMCs suggests a role in organizing the formation of a mature vasculature. It
is very likely to involve interaction with the Notch ligand Jagged1 [7], which is expressed on ECs and VSMCs in the theca layer of growing follicles [4]. It remains unclear as to why we were unable to detect Notch3 in GCs as described
by Johnson et al. [2]. Notch2 was consistently expressed in GCs of preantral and small antral follicles
and sporadic Notch2 staining was also seen in preovulatory follicles. These findings
suggest that Notch2 in GCs is activated by neighbouring GCs expressing Jagged2 [2], although we did not specifically stain for this protein. Our findings confirm the
localization noted in in vitro models [3]. Dll4 is exclusively expressed on ECs. Based on previous results [4,5] and consistent with our data, this suggests that Dll4 expressed on ECs signals to
a neighboring EC expressing Notch1 and possibly Notch4. As Jagged1 is present on ECs,
it might not only signal to VSMCs Notch1/Notch3, but also compete with Dll4 regarding
the interaction with the Notch1 receptor located on neighboring ECs, as suggested
previously by Benedito [13].

Inhibition of Notch function with the γ-secretase inhibitor compound E significantly
blocked gonadotropin-dependent follicle growth up to the preovulatory stage of development.
Thus, the number of follicles evolving to the preovulatory stage was significantly
decreased. Due to the blockage of gonadotropin-dependent follicle development, the
following secondary effects were seen: 1) lesser degree of increase in ovarian weight
due to the inability to develop tertiary follicles similar in number to control; 2)
lesser degree of increase in uterine weight due to lower E2 secretion in the treatment
group when compared to control. In contrast to the effects of VEGF receptor 2 (VEGFR-2)
BAb on gonadotropin-dependent folliculogenesis [9], no reduction in follicular or interstitial area blood vessels is seen in ovaries
subjected to compound E. Even though we did not quantify ECs or VSMCs, our visual
inspection suggests that there might be a slight increase of these cell types in the
treatment group. This supports the finding that vascular cell proliferation continued
to occur at least at a level similar to control in the ovaries from compound E treated
animals.

The salient feature of ovarian vasculature exposed to a γ-secretase inhibitor is its
disorganized appearance. One has the impression that ECs and VSMCs have lost the ability
to connect in an orderly fashion during angiogenesis. These observations may suggest
that compound E induced perturbation of angiogenesis did not allow proper assembly
of blood vessels.

It is of high interest that disruption of EC signaling through YW152F, an anti-Dll4
BAb [11] did not disrupt follicle growth to the preovulatory stage, nor did it affect ovarian
or uterine weight or E2 production or secretion. The blocking of EC Notch1 activation
seems to cause a mild level of disorganization of the interaction of ECs and VSMCs,
but it is insufficient to block functional vascular growth and blood circulation to
support follicle development to the preovulatory stage. In the retina, YW152F creates
a phenotype of non-productive sprouting angiogenesis [11], which is very similar to the effects seen with γ-secretase inhbitors.

The weakness of our YW152F experiment is that one could argue that the absence of
inhibiting effect on folliculogenesis in the treated animals might be due to ineffectiveness
of the administered Dll4 BAb. Unlike with compound E, where the effect can be validated
by observing goblet cell proliferation in the gut, there is no such readily available
positive control for the YW152F treated animals. However, when administering YW152F
during corpus luteum formation in the same animal model, there are profound differences
in angiogenesis when Dll4 is blocked [14]. This can indirectly serve as a proof of action and suggests that different types
of angiogenic development and growth occur in follicular and luteal phase, indicating
that circular elongation angiogenesis observed during follicular growth is quite different
from sprouting angiogenesis in other tissues.

As Notch function is complex, several possibilities exist to explain our results at
the molecular level.

Notch and angiogenesis

During inhibition of Notch function, through compound E or YW152F, PMSG driven VEGF
production in GCs is maintained to stimulate vascular growth by activation of VEGFR-2
on endothelial cells [9,15,16]. Disruption of endothelial Notch1 signaling through blockage of Dll4 is not sufficient
to disrupt coordination of vascular growth in a significant way. In contrast, interference
with Notch1 signaling on endothelial cells, as well as Notch1 and Notch3 signaling
on VSMCs in compound E treated animals disrupts critical coordination between these
2 cell types, which is necessary to form mature functional vasculature required for
gonadotropin-dependent follicular growth. These observations suggest that Notch1 and
Notch3 coordinate VEGF driven angiogenesis in the theca layer during gonadotropin-dependent
folliculogenesis.

Effects of notch on non-angiogenic cells in the ovary

In-situ hybridization studies demonstrate that Notch2 and Notch3 are expressed on
GCs [2]. We did not detect Notch3 on GCs, but did see Notch2 expressed. Johnson et al. speculated
that GCs Notch activity is necessary for proliferation and differentiation, while
preventing follicular atresia due to apoptosis. In vitro models have shown that inhibition of Notch2 leads to reduction of c-Myc inhibiting
granulosa proliferation. Therefore, we suggest that blockage of Notch2 through administration
of compound E might have affected GCs proliferation and differentiation, which in
turn could have contributed to the inhibition of follicle development. In this case,
the absence of significant effects observed in YW152F treated animals would be plausible,
since our immunohistochemistry stains did not demonstrate presence of Dll4 or Notch3
within the follicle and blocking this pathway may have no impact on notch signaling
among granulosa cells. Thus, further experiments with specific inhibition of Notch2
and Jagged2 are needed.

Conclusions

In summary, we demonstrated by immunohistochemistry that members of the Notch family
are expressed primarily in the vasculature (except Notch2) of follicles during folliculogenesis
to the preovulatory stage, and therefore represent a new group of intraovarian regulators.
Among intraovarian regulators, Notch is unique as the ligand and receptor are single-pass
transmembrane proteins, which restricts the Notch pathway to signaling to neighboring
cells [7]. Through functional studies we demonstrated that compound E, a pan-Notch inhibitor,
but not YW152F, a Dll4 blocking antibody, disrupts the assembly of theca layer ECs
with VSMCs enough to diminish gonadotropin-dependent follicle growth. It is meaningful
that this type of vascular disturbance is distinctly different from the non-productive
sprouting angiogenesis seen in the retina when exposed to γ-secretase inhibitors.
It is likely that non-angiogenic Notch2 detected on GCs also plays a role in gonadotropin-dependent
folliculogenesis. Our results represent a preliminary attempt to determine that vascular
and possibly non-vascular Notch play an important role during gonadotropin-dependent
follicle growth to the preovulatory stage of development.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

VPJ and CMS carried out all laboratory experiments, analyzed the data and interpreted
the results. VPJ and RCZ drafted the manuscript edited by CMS and VMS. CJS assisted
in data analysis and optimization of immunohistochemistry. RG performed BrdU staining
and assisted in animal experiments. XW assisted in preparation and administration
of Compound E solution. JK provided material support and participated in design and
coordination. RCZ conceived and implemented the study design. All authors read and
approved the final manuscript.

Acknowledgments

We would like to thank Dr. M. Jan for providing us with the anti-Dll4 YW152F antibody
(Genentech, San Francisco, CA).

References

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